The Ras pathway is an important route of cellular signal transduction, functioning to relay vital signals that control cell growth, proliferation, and differentiation. The Ras proteins themselves are prominent drivers of human cancer, with almost a third of tumors harboring mutant RAS alleles, and mutations in various components of the Ras pathway contribute to a group of developmental disorders termed the RASopathies. Elucidating the molecular mechanisms that regulate Ras pathway signaling and identifying strategies to disrupt signal transmission in human disease states is a major health priority and has been the focus of our laboratory's efforts for almost 30 years. Much of our research has centered on the Raf protein kinases, which are direct effectors of Ras. A primary contribution of our work to the field has been our identification and characterization of key protein interactions and phosphorylation events that modulate Raf function. Early studies from our group were the first to identify a mutation in the Raf kinases that disrupts Ras binding, providing researchers with a key tool to investigate the functional significance of the Ras/Raf interaction. In addition, our work analyzing Raf phosphorylation led to the discovery of inhibitory feedback phosphorylation loops that can impact the effectiveness of certain cancer therapies and are critical for the downregulation of Ras signaling under normal growth conditions and during cellular stress. Our studies demonstrating the role of Raf dimerization have also had important implications for cancer treatment, revealing how disease progression can be altered by secondary mutations or inhibitor treatments that promote Raf dimer formation. Moreover, these studies provided the proof-of-principle that inhibiting Raf dimerization has therapeutic potential. Realizing the importance of studying signaling events under live cell conditions, our group has recently developed bioluminescence resonance energy transfer (BRET) technologies for analyzing Raf regulatory interactions (Ras/Raf binding and Raf dimerization) in live cells. The advantage of the BRET system is that it allows for crucial signaling interactions to be monitored in the context of the plasma membrane environment and under conditions where post-translational modifications still occur, events that can strongly influence protein binding as well as signal progression. Our BRET assay monitoring Raf dimerization was used in a collaborative project with Dr. Craig Ceol's group (U. Mass Worchester) to evaluate the signaling cross-talk between the KIT receptor tyrosine kinase (RTK) and B-Raf V600E in malignant melanomas. These studies revealed that activation of WT-B-Raf induced by KIT signaling could interfere with melanoma formation driven by B-Raf V600E. In collaboration with the NCI-Molecular Target Program (MTP), we are also using the BRET assay to conduct high-throughput screens to identify natural product compounds that can disrupt or prevent Ras/Raf binding or Raf dimerization in live cells. Moreover, the BRET assay has proven to be a very sensitive way of detecting kinase inhibitors that have the deleterious effect of augmenting Ras/Raf binding and promoting paradoxical ERK activation. During this review period, we have further utilized the BRET assay to investigate the Ras/Raf interaction and our studies have revealed pronounced binding preferences between the individual Ras and Raf family members. Our results indicate that C-Raf binds all mutant Ras proteins with high affinity, whereas B-Raf exhibits a striking preference for mutant K-Ras. This selectivity was mediated by the acidic, N-terminal segment of B-Raf and required the polybasic region (PBR) of the K-Ras hypervariable region (HVR) for high-affinity binding. Moreover, through depletion studies, we found that C-Raf is critical for mutant H-Ras-driven signaling and that events promoting stable B-Raf/C-Raf dimer formation, such as certain B-Raf mutations or Raf inhibitor treatments, can allow mutant H-Ras to engage B-Raf with increased affinity to promote tumorigenesis. Together, these findings have revealed a previously unappreciated role for C-Raf in potentiating B-Raf function. In addition, our group has worked in collaboration with colleagues in the NCI-MTP to determine the mechanism by which certain new compounds isolated from their natural product extracts selectively inhibit the growth of tumor lines expressing B-Raf V600E. As a result of this effort, novel madecassic acid derivatives were identified that could inhibit ERK activation in B-Raf V600E-expressing tumor cells by selectively reducing Raf protein levels. In a second study, two newly identified macrophilone-type pyrroloiminoquines isolated from the marine hydroid Macrorhynchia were found to have a similar but more dramatic effect on Raf protein levels in tumor lines expressing B-Raf V600E, indicating that the chemical scaffold of the macrophilones could provide small-molecule therapeutic leads for targeting the Rafs in cancers driven by B-Raf V600E.